107.fm

Size: px

Start display at page:

Download "107.fm"

상아 영
5 years ago
Views:

Nabion Corporation, Seoul, Korea 2 Department of Chemical Engineering, Hanyang University, Seoul, Korea 3 Department of Applied Chemistry, Dongduk Women s University, Seoul, Korea 4 Department of

1 Lab. Anim. Res. 2010: 26(1), Inhibitory Effect of Luteolin Liposome Solution by Animal Model for Atopic Dermatitis in NC/Nga Mice Moon-Jae Choi 1,2, Young-Moo Lee 2, Byung-Suk Jin 3 and Bae-Hwan Kim 4 * 1 Nabion Corporation, Seoul, Korea 2 Department of Chemical Engineering, Hanyang University, Seoul, Korea 3 Department of Applied Chemistry, Dongduk Women s University, Seoul, Korea 4 Department of Public Health, Keimyung University, Daegu, Korea Atopic dermatitis is a common chronic inflammatory skin disease, associated with marked inflammatory cells (of mast cells and eosinophils) and severe itching, which leads to clinical problems in the pediatric population. This study was designed to investigate the inhibitory effects of luteolin liposome solution, that is entrapped the hydrophobic luteolin (one of the flavonoids) into ethosome to improve its stability, by using hapten-induced atopic dermatitis animal model (NC/Nga mice).the luteolin liposome treated mice showed anti-inflammatory effect as evidenced by the lowering of erythema and edema in clinical observation, reduction of inflammatory cell infiltration and epidermal thickness in histopathological examination, when compared with TNCB induced controls. Luteolin liposome solution also reduced the serum IgE level which played important roles in the atopic dermatitis model. These results suggest that luteolin liposome solution has some merit in this formulation showing inhibitory effects for the atopic dermatitis. Key words: Atopic dermatitis, animal model, TNCB, luteolin liposome (Received 21 January 2010; Revised version received 15 February 2010; Accepted 13 March 2010) mv v w w w w y xü» š, 20%, 1-3% ƒ wš, ƒwš y (Schultz-Larsen and Hanifin, 2002; McGirt and Beck, 2006). w w», w mv v e w w e ƒ š (Furue et al., 2004; Schuller et al., 2004; Wisselink and Willemse, 2009), w w v w» š. w z mv v e w w sƒ ã w š. mv v w NC/ Nga w (spontaneously developed *Corresponding author: Bae-Hwan Kim, Department of Public Health, Keimyung University, 2800 Dalgubeoldaero, Dalseo-Gu, Daegu, , Korea Tel: Fax: kim9399@kmu.ac.kr dermatitis) wš, hapten sw š w š (Matsuda et al., 1997; Taniguchi et al., 2003; Yano et al., 2003). NC/Nga mv w w (Shiohara et al., 2004), ûš(50% w), r w» (Matsumoto et al., 1999; Shiohara et al., 2004), w» w hapten mite e e» w mv v x x ùkù w ƒ ü w š (Yun et al., 2008) l (luteolin; 3',4',5,7-tetrahydroxyflavone), w s r w w y, w, w z ƒ š š w w w t, t, y t y w ƒ w š (Chen et al., 2007; Ashokkumar and Sudhandiran, 2008; Kaefer and Milner, 2008). ù l w 47

2 48 Moon-Jae Choi et al. y w š w y û š w w w» w ethosome w s y w. Ethosome v n k w g carrier, k carrier š x w û š ƒ d v û w w š (Touitou et al., 1998; 2000). x NC/Nga hapten w w mv v, l s v sw mv v yw y w. lò Õ 7 f NC/Nga p (» û) l w, 7 k x w. x y 23±3 o C, 40-60%,» 12 w, x (u ùg ) ( ³ ) w œ w. x y 3R x z(institutional Animal Care and Use Committee, IACUC) e wš d š w ww. l FUIPTPNF Ethosome HPC (hydrogenated phosphatidyl choline) l (Wilshire Tech) 9:1 yw 1g k 1mL wì v j z š 60 o C ƒ w z, k k 1mL š 2 j y (hydrated liquid crystal) x g ( y ). z» k ƒw rv 60 ml 5mL/min w ƒw y ù j» carrier xk w ethosome. w 10 ƒ ww z v j bath type sonicator 10 q ƒw z 1 j þƒ g. l 7 z,» x 5 4 ù. x, x n ú w, 3 mv v šw (Taniguchi et al., 2003), 5% TNCB (1-Chloro-2,4,6-trinitrobenzene, Aldrich) 150 µl 1z sw z, 2 l 1% TNCB 1z 5 mv v w. TNCB (1%) sw z, T1 l s, T2 l PEG % ƒ 150 µl 5z 2 v sw, TNCB NC( ) w. ²n x» 1z w. x w» w, e ú eg w 30 z w z w. w w w grooming w (Carstens and Kuraishi, 2004), w y ² i w (facial scratching) wš w mv v w (hindlimb scratching) w z d w. j TNCB e x v d e Tewameter (TM300, CK electronic GmbH, Germany) w TEWL (transepidermal water loss) d w, Corneometer (CM825, CK electronic GmbH, Germany) w w d w w. j j á x n w v v (, x,, y, ƒvkv ) ¾ w š, v w 10% neutral formalin š wš k k z paraffin s w r w. r H&E w tv v v w, tv Ì w. w toluidine blue mw s k w. l *H& )JTUBNJOF Th2 cytokine w B cell w x ü IgE level (Renz et al., 1994), s rw sƒw mv v x q w w tƒ d w. x ü IgE w Enzyme-Linked Immunosorbent (ELISA) Assay w x ƒ isoflurane w

3 Atopic inhibitory effect of Luteolin liposome 49 w z l x wš» 3,000 rpm 10 w x w, 20 o C w w. IgE d sandwich ELISA w w (SHIBAYAGI ELISA kit, Japan). m SPSS 17.0 for windows (SPSS Inc., USA) w one-way ANOVA w š ƒ w» w Duncan's multiple range test w z w š, m P<0.05 w. ²n x» x» w y y w (data not shown). v z (NC) w TNCB ƒ ƒ š(p<0.05), x e w w (Table 1). j TEWL e x e T1 (Luteolin Liposome solution) T2 (Luteolin) e (0 week) w 2 z (P<0.01), TNCB w (P<0.01) z w j e q, x d TNCB w x e T1 T2 w ù w w (Table 2). Corneometer w w d, T1 (Luteolin Liposome solution) T2 (Luteolin) (NC) w û (P<0.05), TNCB w (Table 3). j j á e TNCB y, ƒvkv w x ¾ mv v w v ùkü š, T1 (Luteolin Liposome solution) z, T2 (Luteolin) û (Figure 1). t tv Ì(epidermal hyperplasia, hyperkeratosis) TNCB w ƒƒ š, T1 (Luteolin Liposome solution) T2 (Luteolin) TNCB w v ü š. w se TNCB w T1 T2 z (Table 4, Figure 2). v ü s(mast cell) TNCB (NC) w ƒƒ š, TNCB w x e T1 (Luteolin Liposome solution) T2 (Luteolin) Table 1. The frequency of scratching in NC/Nga mice 0 0.8±0.8 a 6.0±2.6 b 5.2±2.6 b 5.2±2.2 b 2 0.6±0.5 a 4.4±2.1 b 2.6±1.5 ab 2.4±1.7 ab Unit: times in 30 minutes NC: Normal control, TNCB: TNCB induced control, T1: Luteolin liposome solution, T2: Luteolin Values with different superscripts (a,b) in the same row are significantly different (P<0.05) by ANOVA and Duncan s multiple range test. Table 2. Changes inf transepidermal water loss (TEWL) of NC/Nga mice ±2.93 a 75.04±13.89 b 80.28±15.58 b 76.44±27.05 b ±3.78 a 41.68±12.78 b 38.44±20.27 b ±22.51 ab Unit: g/m²/h Values with different superscripts (a,b) in the same row are significantly different (P<0.05) by ANOVA and Duncan s multiple range test. Table 3. Changes in water capacity of NC/Nga mice by Corneometer ±25.79 b 16.86±2.93 a 14.01±7.27 a 16.74±5.04 a ±4.32 b ±11.22 a 19.86±4.37 a 20.23±7.19 a Unit: Arbitrary unit Values with different superscripts (a,b) in the same row are significantly different (P<0.05) by ANOVA and Duncan's multiple range test.

50 Moon-Jae Choi et al. Figure 1. Gross lesions of NC/Nga mouse skin. A: Normal control, B: TNCB induced control, C: Luteolin liposome solution, D: Luteolin. ƒ. k w (Table 5, Figure 3).

4 50 Moon-Jae Choi et al. Figure 1. Gross lesions of NC/Nga mouse skin. A: Normal control, B: TNCB induced control, C: Luteolin liposome solution, D: Luteolin. ƒ. k w (Table 5, Figure 3). l *H& x mv v t w t x ü IgE d w TNCB w m v ƒƒ, x e T1 (Luteolin Liposome solution) T2 (Luteolin) x ü IgE level TNCB w (Table 6). mv v w z y sƒw» w sƒ kw w. z sƒ mv v in vivo x» x ³ w w, x w. p š NC/Nga v x, NC/Nga hapten e w j mv v x ƒÿ š (Shiohara et al., 2004). mv v e z sƒ ƒ hapten w z sƒw. hapten DNCB (2,4-dinitrochlorobenzene) (Lee et al., 2006)ù TNCB (Taniguchi et al., 2003)ƒ š, sƒ TNCB w. hapten v v w, n z, š n Th1 w v w ù mv v p Th2» w ƒ š (Plitnick et al., 2002; Taniguchi et al., 2003; Goutet et al., 2005; Cumberbatch et al., 2005). Table 4. Epidermal thickness of NC/Nga mice ±6.02 a ±43.37 c 34.87±10.91 ab 52.74±17.76 b Unit: µm Epidermal thickness was determined by the average of three sites (short, medium, long-size each) in same fields of microscope. Values with different superscripts (a,b,c) in the same row are significantly different (P<0.05) by ANOVA and Duncan s multiple range test.

5 Atopic inhibitory effect of Luteolin liposome 51 Figure 2. Histological observation of NC/Nga mouse skin by H&E staining ( 100) (red bar=50 µm). A: Normal control, B: TNCB induced control, C: Luteolin liposome solution, D: Luteolin. mv v hapten n, z, Th1 y Th2 e e š š (Mueller et al., 1989; Goutet et al., 2005; Friedman, 2006), TNCB n z» y w mv w w š w. mv v v v y w, w epidermal hyperplasia v d ü s e š, s v ƒ y w. p» (Renz et al., 1994) w mv ƒ w t B s w x IgE e ƒ Th2 w x w mv v z sƒ ww x š w. mv v ƒ x IgE e Table 5. Number of mast cells and ratio of degranulation by Toluidine blue staining x (Luteolin Liposome solution Luteolin) s w w. mv ƒ x ü IgE v ü s(mast cell) k g histamine w w (Falus Merétey, 1992; Malaviya et al., 1996; Menzies-Gow et al., 2002; Ying et al., 1999) x IgE e k» wš. ƒ w vü s w» q. x tv (TEWL) v ü w d w, v sƒw t (Elias and Menon, 1991; Hruza and Pentland, 1993). x ƒ w TNCB w NC TNCB T1 T2 Total No of mast cell 79.2±11.5 a 176.6±17.8 c 134.0±26.1 b 125.3±37.8 b Mean No of degranulation.59±8.6 a 0.117±12.9 b 101.6±22.9 b 00.94±28.4 b Deganulation/total No The number of mast cells was determined by the average of five different fields with an eyepiece of 100 magnification. Values with different superscripts (a,b,c) in the same row are significantly different (P<0.05) by ANOVA and Duncan s multiple range test.

6 52 Moon-Jae Choi et al. Figure 3. Histological observation of NC/Nga mouse skin by Toluidine blue staining ( 100) (red bar=50 µm). A: Normal control, B: TNCB induced control, C: Luteolin liposome solution, D: Luteolin. There are many mast cells (arrow) in TNCB control (B). w ù j. l (luteolin) w w y, w, w ƒ s r (Chen et al., 2007; Ashokkumar and Sudhandiran, 2008; Kaefer and Milner, 2008) mv e z» w q š û x ethosome w s y mw z w. x s y l» l mv v t x ü IgE w w w š, t { œ q. x Luteolin Liposome solution TNCB mv TNCB w t v w w w v q š, p x ü IgE level v vü s w mv v z ƒ. k Ashokkumar, P. and Sudhandiran, G. (2008) Protective role of luteolin on the status of lipid peroxidation and antioxidant defense against azoxymethane-induced experimental colon carcinogenesis. Biomed. & Pharmacotherapy 62, Carstens, E. and Kuraishi, Y. (2004) Animal models of itch: scratching away at the problem. In Itch Basic Mechanisms and Therapy (Yosipovitch, G. ed.), pp.35-50, Informa Healthcare, USA. Chen, C.Y., Peng, W.H., Tsai, K.D. and Hsu, S.L. (2007) Luteolin suppresses inflammation-associated gene expression by blocking NF-κB and AP-1 activation pathway in mouse alveolar macrophages. Life Sci. 81, 1602Æ1614. Cumberbatch, M., Clelland, K., Dearman, R.J. and Kimber, I. (2005) Impact of cutaneous IL-10 on resident epidermal Langerhans' cells and the development of polarized immune responses. J. Immunol. 175(1), Elias, P.M. and Menon, G.X. (1991) Structural and lipid biochemical correlation of the epidermal permeability barrier. Table 6. Plasma IgE levels of NC/Nga mice ±364.6 a 4,303.3±523.0 c ±780.5 b 2,503.2±1,041.5 b Unit: ng/ml Values with different superscripts (a,b,c) in the same row are significantly different (P<0.05) by ANOVA and Duncan s multiple range test.

7 Atopic inhibitory effect of Luteolin liposome 53 Adv. Lipid Res. 24, Falus, A. and Merétey, K. (1992) Histamine: an early messenger in inflammatory and immune reactions. Immunol. Today 13(5), Friedman, P.S. (2006) Contact sensitization and allergic contact dermatitis immunobiological mechanisms. Toxicol. Lett. 162, Furue, M., Terao, H., Moroi, Y., Koga, T., Kubota, Y., Nakayama, J., Furukawa, F., Tanaka, Y., Katayama, I., Kinukawa, N., Nose, Y. and Urabe, K. (2004) Dosage and adverse effects of topical tacrolimus and steroids in daily management of atopic dermatitis. J. Dermatol. 31(4), Goutet, M., Pépin, E., Langonné, I., Huguet, N. and Ban, M. (2005) Identification of contact and respiratory sensitizers using flow cytometry. Toxicol. Appl. Pharmacol. 205(3), Hruza, L.L. and Pentland, A.P. (1993) Mechanism of UV induced inflammation. J. Invest. Dermatol. 15(2), Kaefer, C.M. and Milner, J.A. (2008) The role of herbs and spices in cancer prevention. J. Nutri. Biochem. 19, Lee, S.H., Baek, S.J., Kim, H.A. and Heo, Y. (2006) 2,4- dinitrochlorobenzene-induced atopic dermatitis like immune alteration in mice. J. Toxicol. Pub. Health 22(4), Malaviya, R., Morrison, A.R. and Pentland, A.P. (1996) Histamine in human epidermal cells is induced by ultraviolet light injury. J. Invest. Dermatol. 106(4), Matsuda, H., Watanabe, N., Geba, G.P., Sperl, J., Tsudzuki, M., Hiroi, J., Matsumoto, M., Ushio, H., Saito, S., Askenase, P.W. and Ra, C. (1997) Development of atopic dermatitis-like skin lesion with IgE hyperproduction in NC/Nga mice. Int. Immunol. 9(3), Matsumoto, M., Ra, C., Kawamoto, K., Sato, H., Itakura, A., Sawada, J., Ushio, H., Suto, H., Mitsuishi, K., Hikasa, Y. and Matsuda, H. (1999) IgE hyperproduction through enhanced tyrosine phosphorylation of Janus kinase 3 in NC/Nga mice, a model for human atopic dermatitis. J. Immunol. 162(2), McGirt, L.Y. and Beck L.A. (2006) Innate immune defects in atopic dermatitis. J. of Allergy and Clin. Immunol. 118(1), Menzies-Gow, A., Ying, S., Sabroe, I., Stubbs, V.L., Soler, D., Williams, T.J. and Kay, A.B. (2002) Eotaxin (CCL11) and eotaxin-2 (CCL24) induce recruitment of eosinophils, basophils, neutrophils, and macrophages as well as features of early- and late-phase allergic reactions following cutaneous injection in human atopic and nonatopic volunteers. J. Immunol. 169(5), Mueller, D.L., Jenkins, M.K. and Schwartz, R.H. (1989) Clonal expansion versus functional clonal inactivation: a costimulatory signalling pathway determines the outcome of T cell antigen receptor occupancy. Annu. Rev. Immunol. 7, Plitnick, L.M., Loveless, S.E., Ladics, G.S., Holsapple, M.P., Selgrade, M.J., Sailstad, D.M. and Smialowicz, R.J. (2002) Cytokine profiling for chemical sensitizers: application of the ribonuclease protection assay and effect of dose. Toxicol. Appl. Pharmacol. 179(3), Renz, H., Brodie, C., Bradley, K., Leung, D.Y. and Gelfand, E.W. (1994) Enhancement of IgE production by anti-cd40 antibody in atopic dermatitis. J. Allergy Clin. Immunol. 93(3), Schuller, E., Oppel T., Bornhövd, E., Wetzel, S. and Wollenberg, A (2004) Tacrolimus ointment causes inflammatory dendritic epidermal cell depletion but no Langerhans cell apoptosis in patients with atopic dermatitis. J. Allergy and Clin. Immunol. 114(1), Schultz-Larsen, F. and Hanifin, J.M. (2002) Epidemiology of atopic dermatitis. Immunol. Allergy Clin. North Am. 22, Shiohara, T., Hayakawa, J. and Mizukawa, Y. (2004) Animal models for atopic dermatitis: are they relevant to human disease? J. Dermatol. Sci. 36(1), 1-9. Taniguchi, Y., Kohno, K., Inoue, S., Koya-Miyata, S., Okamoto, I., Arai, N., Iwaki, K., Ikeda, M. and Kurimoto, M. (2003) Oral administration of royal jelly inhibits the development of atopic dermatitis-like skin lesions in NC/Nga mice. International Immunopharmacol. 3(9), Touitou, E., Dayan, N., Bergelson, L., Godin, B. and Eliaz, M. (2000) Ethosomes-novel vesicular carriers for enhanced delivery: characterization and skin penetration properties J. Control. Release, 65, Touitou, E., Meidan, V.M. and Horwitz, E. (1998) Methods for quantitative determination of drug localized in the skin. J. Control. Release, 56, Wisselink, M.A. and Willemse T (2009) The efficacy of cyclosporine A in cats with presumed atopic dermatitis: A double blind, randomised prednisolone-controlled study. The Vet. Journal, 180, Yano, S., Umeda, D., Yamashita, S., Yamada, K. and Tachibana, H. (2009) Dietary apigenin attenuates the development of atopic dermatitis-like skin lesions in NC/Nga mice. J. Nutri. Biochem. 20(11), Ying, S., Robinson, D.S., Meng, Q., Barata, L.T., McEuen, A.R., Buckley, M.G., Walls, A.F., Askenase, P.W. and Kay, A.B. (1999) C-C chemokines in allergen-induced late-phase cutaneous responses in atopic subjects: association of eotaxin with early 6-hour eosinophils, and of eotaxin-2 and monocyte chemoattractant protein-4 with the later 24-hour tissue eosinophilia, and relationship to basophils and other C- C chemokines (monocyte chemoattractant protein-3 and RANTES). J. Immunol. 163(7), Yun, J.W., Kim, H., Kang, H.J., Koh, J.Y. and Kim, B.H. (2008): Models for Atopic Dermatitis in NC/Nga Mice. Lab. Animal Research, 24(1),

16(1)-3(국문)(p.40-45).fm

16(1)-3(국문)(p.40-45).fm w wz 16«1y Kor. J. Clin. Pharm., Vol. 16, No. 1. 2006 x w$btf3fqpsu'psn û w m w Department of Statistics, Chonnam National University Eunsik Park College of Natural Sciences, Chonnam National University